Methods and compositions for increasing nuclease activity

ABSTRACT

Methods and compositions for increasing nuclease activity by subjecting cells expressing the nuclease to hypothermic conditions to increase activity of the nucleases for genomic modifications.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication No. 61/216,526, filed May 18, 2009, the disclosure of whichis hereby incorporated by reference in its entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

TECHNICAL FIELD

The present disclosure is in the field of genome engineering,particularly increasing nuclease activity.

BACKGROUND

Nucleases, including zinc finger nucleases and homing endonucleases suchas I-SceI, that are engineered to specifically bind to target sites havebeen shown to be useful in genome engineering in basic research and inthe pharmaceutical and biotechnology applications. For example, zincfinger nucleases (ZFNs) are proteins comprising engineered site-specificzinc fingers (with engineered recognition regions) fused to a nucleasedomain. Such ZFNs have been successfully used for genome modification ina variety of different species. See, for example, United States PatentPublications 20030232410; 20050208489; 20050026157; 20050064474;20060188987; 20060063231; and International Publication WO 07/014,275,the disclosures of which are incorporated by reference in theirentireties for all purposes. These ZFNs can be used to create adouble-strand break (DSB) in a target nucleotide sequence, whichincreases the frequency of donor nucleic acid introduction viahomologous recombination at the targeted locus (targeted integration)more than 1000-fold. In addition, the inaccurate repair of asite-specific DSB by non-homologous end joining (NHEJ) can also resultin gene disruption. Nucleases can be used for a wide variety of purposessuch as for cell line engineering as well as for therapeuticapplications.

Efficiency of nuclease activity can be influenced by a variety offactors such as accessibility of the target and the quality of thebinding interaction between the nuclease and its target nucleic acid. Toincrease the success rate of nuclease driven genomic modifications,researchers often have to resort to introducing selectable markersduring donor integration in order to be able to select variants thathave had modifications from those that have not been modified (see, forexample, U.S. Pat. No. 6,528,313). For a number of applications, use ofselectable markers is not desirable as this technique leaves anadditional gene or nucleic acid sequence inserted into the genome.

Thus, there remains a need for compositions and methods for increasingnuclease activity to allow for more efficient use of these powerfultools.

SUMMARY

Described herein are methods and compositions for increasing activity ofan exogenous nuclease (e.g.; ZFN) in a host cell. The methods involvethe use of transient hypothermia, i.e. “cold shock,” to cells followingtransfection with the expression vectors encoding the nuclease. Thecells are held at the reduced temperature for an extended period of timeand then are shifted back to the appropriate temperature to recover andincrease cell division, a method that can increase the rate of genedisruption by more than ten-fold. The methods and compositions may beused for targeted genomic modification through introduction of mutationsvia NHEJ, and also may be used for targeted donor nucleic acid insertionvia homologous recombination. The methods and compositions describedherein significantly increase the efficiency of nuclease activity in ahost cell.

In one aspect, described herein is a method for increasing activity ofan exogenous nuclease (e.g., in mammalian cells by subjecting the cellsto cold shock following transfection, and, following the cold shock,returning the cells to an appropriate temperature for growth). Incertain embodiments, the cold shock temperature is between 27 and 33° C.In certain embodiments, the cells are subjected to cold shock forbetween 1 and 4 days.

In another aspect, the invention provides a host cell comprising anuclease expression plasmid wherein the host cell has been subject to acold shock. In another aspect, the nuclease(s) is(are) delivered to thecell via Integration Defective Lentiviral (IDLY) constructs (see forexample United States Patent Publication 2009/0117617, incorporatedherein by reference) or by integration competent lentiviral vectors. Inanother aspect, the nuclease(s) is(are) delivered to the cell via anadenoviral vector or an adenoviral associated vector (AAV). In oneaspect, the invention provides a mammalian host cell at 33° C. or lowercomprising a nuclease (e.g., ZFN) expression plasmid and a donor nucleicacid such that the nuclease mediates targeted integration of theexogenous sequence into the genome. In certain embodiments, the cell isa eukaryotic cell (e.g., a mammalian cell). In some aspects, the hostcells are an established cell line while in other aspects, the host cellis a primary cell isolated from a mammal. In some aspects, the inventionprovides a host cell as above wherein the donor nucleic acid encodes areporter construct which may be transiently or stably expressed in thehost cell. Any of the host cells may further comprise a sequenceencoding a nuclease, for example a homing nuclease or zinc fingernuclease.

In another aspect, the invention provides a host cell comprising a donornucleic acid wherein the donor nucleic acid has been integrated into thegenome of the host cell using the methods provided herein. In certainembodiments, the cell is a eukaryotic cell (e.g., a mammalian cell). Anyof the host cells may further comprise a sequence encoding a nuclease,for example a homing nuclease or zinc finger nuclease.

In yet another aspect, provided herein is a method of increasing thenuclease activity of a known nuclease, the method comprising the stepsof: introducing one or more expression constructs that express thenuclease(s) into any of the host cells described herein, incubating thecells under cold shock conditions such that the nuclease is expressedbut the rate of cell division is greatly reduced; and then culturing thecells in an appropriate temperature such that the rate of cell divisionincreases, thereby increasing the nuclease activity of the knownnuclease. In certain embodiments, the methods further comprise the stepof determining the level of nuclease activity. In any of the methodsdescribed herein, the nuclease may comprise, for example, anon-naturally occurring DNA-binding domain (e.g., an engineered zincfinger protein, a TAL-effector nuclease fusion protein, or an engineeredDNA-binding domain from a homing endonuclease). In certain embodiments,the nuclease is a zinc finger nuclease (ZFN) or pair of ZFNs. In otherembodiments, the nuclease is a TAL-effector domain nuclease fusionprotein.

Any of the methods may further comprise introducing an exogenoussequence into the host cell such that the nuclease mediates targetedintegration of the exogenous sequence into the genome. In certainembodiments, the exogenous sequence is introduced at the same time asthe nuclease(s). In some aspects, the exogenous sequence may comprise areporter gene. In certain embodiments, the methods further comprisingisolating the cells expressing the reporter gene. In any of the methodsdescribed herein, the genomic modification is a gene disruption and/or agene addition.

Furthermore, in any of the methods described herein, the nuclease(s)(e.g., ZFN, ZFN pair, TAL-effector domain nuclease fusion protein,engineered homing endonuclease and/or fusion or a naturally occurring orengineered homing endonuclease. DNA-binding domain and heterologouscleavage domain) may be known to recognize the endogenous targetsequence, for example from results obtained from in vitro assayexperiments.

In another aspect, the invention provides kits that are useful forincreasing the activity of nucleases (e.g. ZFNs, TAL-effector domainnuclease fusion proteins, or engineered homing endonucleases). The kitstypically include one or more nucleases that bind to a target site,optional cells containing the target site(s) of the nuclease andinstructions for introducing the nucleases into the cells and coldshocking the cells to increase nuclease activity. In certainembodiments, the kits comprise at least one construct with the targetgene and a known nuclease capable of cleaving within the target gene.Such kits are useful for optimization of cleavage conditions in avariety of varying host cell types. Other kits contemplated by theinvention may include a known nuclease capable of cleaving within aknown target locus within a genome, and may additionally comprise adonor nucleic acid encoding a reporter gene. Such kits are useful foroptimization of conditions for donor integration. In such kits, thereporter gene may be operatively linked to a polyadenylation signaland/or a regulatory element a promoter).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a gel depicting a comparison of ZFN activity as determinedby the CEL-I assay (SURVEYOR™, Transgenomic) at 30° C. versus 37° C.Varying amounts of AAVS1-specific ZFN expression plasmid were used: 0.5,1 or 2.5 μg. K562 cells were transfected with the expression plasmid andthen divided into two populations to recover at either 30° C. or 37° C.for 4 days. At that time, cells were processed for the CEL-I assay todetect any mismatches that have occurred due to NHEJ activity. Thepercent NHEJ activity is indicated at the bottom of each lane. The datademonstrate that the activity of the ZFNs is increased when the cellsare held at 30° C. At observed NHEJ percentages greater thanapproximately 40%, the results from the CEL-I assay become non-linear.Thus NHEJ percentages greater than 40% are estimates and are indicatedwith an asterisk (*).

FIG. 2, panels A and B, depict the effect of cold shock on the sixdifferent ZFNs indicated at the top of each lane. K562 cells weretransfected with 0.4 μg of the indicated ZFN expression vector andincubated for 3 days at 37° C. (FIG. 2A) or 1 day at 37° C. followed by2 days at 30° C. (FIG. 2B). The frequency of small insertions ordeletions (indels) is shown beneath each lane. In all cases, cold shocktreatment increased the ZFN activity, as determined by CEL-1 assay.

FIG. 3, panels A and B, are gels depicting CEL-I assay results of HeLacells transfected with nucleases at 30° C. or 37° C. Cells weretransfected with 0.1, 0.2 and 0.4 μg of ZFN expression constructencoding AAVS1-specific ZFNs, and, following transfection were dividedinto two populations and allowed to recover at either 30° C. or 37° C.for 3 days. FIG. 3A shows the results of the CEL-I assay for NHEJactivity performed at the end of the 3 days and shows that ZFN activitywas greatly increased when the cells were allowed to recover at 30° C.Following the initial 3 day incubation at either 30° C. or 37° C., cellswere all placed at 37° C. for a period of an additional 21 days.Following this incubation at 37° C., cells were processed for the CEL-Iassay as described above. FIG. 3B shows that the increased genomemodification seen in the population of cells initially incubated at 30°C. is stable over an additional 7 days.

FIG. 4, panels A and B, are gels depicting CEL-I assay results of HeLacells transfected with KDR-targeted nucleases at 30° C. or 37° C. Cellswere transfected with 0.1, 0.2 and 0.4 μg of ZFN expression plasmid,and, following transfection were divided into two populations andallowed to recover at either 30° C. or 37° C. for 3 days. FIG. 4A showsthe results of the CEL-I assay for NHEJ activity performed at the end ofthe 3 days and shows that ZFN activity was greatly increased when thecells were allowed to recover at 30° C. Following the initial 3 dayincubation at either 30° C. or 37° C., cells were all placed at 37° C.for a period of an additional 7 days. Following this 7 day incubation at37° C., cells were processed for the CEL-I assay as described above.FIG. 4B shows that the increased genome modification seen in thepopulation of cells initially incubated at 30° C. is stable over 21 celldoublings under normal growth conditions (i.e. 37° C.). In particular,up to ˜25% of the chromatids were modified in the cold shock-treatedpopulation as compared to 1% in cells incubated at 37° C.

FIG. 5, panels A and F, show preferential cleavage of the ZFN targetsite under cold shock conditions. FIGS. 5A and 5B show results of K652cells nucleofected with a GFP expression plasmid (−) or 80 ng of a CMVpromoter-driver ZFN expression vector (lanes 2-5 and 7-10) targeted tothe GR gene containing either wild-type Fold cleavage domains orobligate heterodimer Fold cleavage domains. Immediately aftertransfection, cells were divided and incubated for 3 days at 37° C.(lanes 2-3 and 7-8) or 3 days at 30° C. (lanes 4-5 and 9-10). Theheterodimeric variants (lanes 2-5) and wild-type FokI domain (lanes7-10) were compared. The frequency of indels at the GR locus wasassessed by CEL-1 assay 3 days post-transfection. FIGS. 5C and 5D showthe frequency of indels in intron 1 of Trim26 (off-target) as assessedby CEL-1 assay 3 days post-transfection of the GR-targeted ZFNsdescribed above. FIGS. 5E and 5F show the frequency of indels in intron1 of chromosome 1 (off-target) as assessed by CEL-1 assay 3 dayspost-transfection of the GR-targeted ZFNs described above.

DETAILED DESCRIPTION

Described herein are compositions and methods to increase nucleaseactivity and kits comprising the methods described. In particular, themethods use transient hypothermia for varying length of times followinghost cell transfection with the nuclease expression plasmid(s). Afterthe period of cold shock, the host cells are returned to a moreappropriate temperature to allow the cells to initiate or increase celldivision. In addition, the compositions and methods described herein canalso be used to optimize nuclease cleavage conditions for genedisruption and/or gene addition in a variety of host cells.

Engineered nuclease technology is based on the engineering of naturallyoccurring DNA-binding proteins. For example, engineering of homingendonucleases with tailored DNA-binding specificities has beendescribed. Chames et al. (2005) Nucleic Acids Res 33(20):e178; Arnouldet al. (2006) J. Mol. Biol. 355:443-458. In addition, engineering ofZFPs has also been described. See, e.g., U.S. Pat. Nos. 6,534,261;6,607,882; 6,824,978; 6,979,539; 6,933,113; 7,163,824; and 7,013,219.

In addition, ZFPs have been fused to nuclease domains to create ZFNs—afunctional entity that is able to recognize its intended nucleic acidtarget through its engineered (ZFP) DNA binding domain and cause the DNAto be cut near the ZFP binding site via the nuclease activity. See,e.g., Kim et al. (1996) Proc Natl Acad Sci USA 93(3):1156-1160. Morerecently, ZFNs have been used for genome modification in a variety oforganisms. See, for example, United States Patent Publications20030232410; 20050208489; 20050026157; 20050064474; 20060188987;20060063231; and International Publication WO 07/014,275.

The biological activity of nucleases is not always the same from celltype to cell type. Thus, methods which can increase nuclease activitycan be used to increase the success rate in a variety of cell types foreither targeted gene disruption at a specified locus throughnuclease-mediated NHEJ, or to increase the amount of geneaddition/deletion through nuclease-mediated homologous recombination.

Thus, the methods and compositions described herein provide highlyefficient and rapid methods for increasing biological activity ofnucleases in vivo. The methods and compositions described herein alsoprovide the components for kits to allow for optimization andcharacterization of nucleases within a cell.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

Definitions

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

Zinc finger binding domains (e.g., the recognition helix region) can be“engineered” to bind to a predetermined nucleotide sequence. Theengineered region of the zinc finger is typically the recognition helix,particularly the portion of the alpha-helical region numbered −1 to +6.Backbone sequences for an engineered recognition helix are known in theart. See, e.g., Miller et al. (2007) Nat Biotechnol 25, 778-785.Non-limiting examples of methods for engineering zinc finger proteinsare design and selection. A designed zinc finger protein is a proteinnot occurring in nature whose design/composition results principallyfrom rational criteria. Rational criteria for design include applicationof substitution rules and computerized algorithms for processinginformation in a database storing information of existing ZFP designsand binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242;and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO02/016536 and WO 03/016496.

A “selected” zinc finger protein is a protein not found in nature whoseproduction results primarily from an empirical process such as phagedisplay, interaction trap or hybrid selection. See e.g., U.S. Pat. No.5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat. No. 6,007,988; U.S. Pat.No. 6,013,453; U.S. Pat. No. 6,200,759; WO 95/19431; WO 96/06166; WO98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO 01/88197 and WO02/099084.

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The terms “first and second cleavage half-domains;” “+ and −cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Patent Publication Nos. 2005/0064474, 20070218528 and 2008/0131962,incorporated herein by reference in their entireties.

“Cold shock” refers to a shift in temperature wherein cells are placedin a hypothermic environment that is colder than optimal growthtemperature. The cold shock temperature will depend on the cell type, inparticular the temperature that is optimal for cell division to occur inthat cell type. For mammalian cells, cold shock temperatures willtypically be, 33° C., 32° C., 31° C., 30° C., 29° C., and 28° C. or evenlower. Zebrafish cell lines are grown at 28° C., so an optimal coldshock temperature would be lower than 28° C., for example, lower than25° C., 24° C., 23° C., 22° C., or even lower. Similarly, plantprotoplasts divide at cooler temperatures than mammalian cells, and so asuitable cold shock temperature would necessarily be cooler than thatused for mammalian cells.

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist. For example, thesequence 5′-GAATTC-3′ is a target site for the Eco RI restrictionendonuclease.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein; polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can, be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPDNA-binding domain and a cleavage domain) and fusion nucleic acids (forexample, a nucleic acid encoding the fusion protein described supra).Examples of the second type of fusion molecule include, but are notlimited to, a fusion between a triplex-forming nucleic acid and apolypeptide, and a fusion between a minor groove binder and a nucleicacid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a, protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

“Eukaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells(e.g., T-cells).

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFPDNA-binding domain is fused to a cleavage domain, the ZFP DNA-bindingdomain and the cleavage domain are in operative linkage if, in thefusion polypeptide, the ZFP DNA-binding domain portion is able to bindits target site and/or its binding site, while the cleavage domain isable to cleave DNA in the vicinity of the target site.

A “vector” is capable of transferring gene sequences to target cells.Typically, “vector construct,” “expression vector,” and “gene transfervector,” mean any nucleic acid construct capable of directing theexpression of a gene of interest and which can transfer gene sequencesto target cells. Thus, the term includes cloning, and expressionvehicles, as well as integrating vectors.

A “reporter gene” or “reporter sequence” refers to any sequence thatproduces a protein product that is easily measured, preferably althoughnot necessarily in a routine assay. Suitable reporter genes include, butare not limited to, sequences encoding proteins that mediate antibioticresistance (e.g., ampicillin resistance, neomycin resistance, G418resistance, puromycin resistance), sequences encoding colored orfluorescent or luminescent proteins (e.g., green fluorescent protein,enhanced green fluorescent protein, red fluorescent protein,luciferase), and proteins which mediate enhanced cell growth and/or geneamplification (e.g., dihydrofolate reductase). Epitope tags include, forexample, one or more copies of FLAG, His, myc, Tap, HA or any detectableamino acid sequence.

Overview

Described herein are compositions and methods for increasing thebiological activity of nucleases within a cell. The compositions andmethods described are effective in increasing nuclease activity in avariety of cell types wherein a desired genomic modification is needed.In the methods described herein, nucleic acids encoding a nuclease(s)specific for a desired target site(s) are introduced into a host cell.Following introduction of the nuclease-encoding nucleic acid, the cellsare subject to a period of “cold shock” by placing the transfected cellsin a hypothermic environment for a period of time. During the period ofcold shock time, nucleases are expressed and are active but the abilityof the host cells to divide is reduced or eliminated. Following theperiod of cold shock, the cells are returned to a temperature thatincreases the rate of cell division. The transient period of cold shockunexpectedly increases the efficiency of nuclease activity with aconcomitant increase in either stimulated homologous recombination inthe presence of a donor nucleic acid, or an increase in imprecisenon-homologous end joining (NHEJ), without any observed deleteriouseffects.

Thus, described herein are rapid and efficient methods for increasingbiological activity of nucleases. The methods have applications in awide variety of cell types. Accordingly, the compositions and methodsdescribed herein can also be utilized in kits that allow the user toscreen nucleases and to select cells with desired genomic modifications.The methods and compositions can also be used to facilitate theisolation of knock-out cell lines because the efficiency of nucleasedigestion is greatly increased. This technology has application in thecreation of cells and/or transgenic organisms that exhibit ‘traitstacking’ due to its ability to increase genome modification efficiency.The invention may also be used to increase the therapeutic applicationsof nucleases since it increases activity in a variety of cell types.

Host Cells

Any host cell wherein a genomic modification is desired may be used withthe practice of the present disclosure. The cell types can be cell linesor natural (e.g., isolated) cells such as, for example, primary cells.Cell lines are available, for example from the American Type CultureCollection (ATCC), or can be generated by methods known in the art, asdescribed for example in Freshney et al., Culture of Animal Cells, AManual of Basic Technique, 3rd ed., 1994, and references cited therein.Similarly, cells can be isolated by methods known in the art. Othernon-limiting examples of cell types include cells that have or aresubject to pathologies, such as cancerous cells and transformed cells,pathogenically infected cells, stem cells, fully differentiated cells,partially differentiated cells, immortalized cells and the like.Prokaryotic (e.g., bacterial) or eukaryotic (e.g., yeast, plant, fungal,piscine and mammalian cells such as feline, canine, murine, bovine,porcine and human) cells can be used, with eukaryotic cells beingpreferred. Suitable mammalian cell lines include K562 cells, CHO(Chinese hamster ovary) cells, 293 cells, HEP-G2 cells, BaF-3 cells,Schneider cells, COS cells (monkey kidney cells expressing SV40T-antigen), CV-1 cells, HuTu80 cells, NTERA2 cells, NB4 cells, HL-60cells and HeLa cells, 293 cells (see, e.g., Graham et al. (1977) J. Gen.Virol. 36:59), and myeloma cells like SP2 or NS0 (see, e.g., Galfre andMilstein (1981) Meth. Enzymol. 73(B):3 46), rat C6 cells, and porcinePk15 cells. Peripheral blood mononucleocytes (PBMCs) or T-cells can alsoserve as hosts. Additionally, suitable cells for use with the inventioninclude stem cells such as primary stem cells as well as inducedpluripotent stem cells. Other eukaryotic cells include, for example,insect (e.g., sp. frugiperda), fungal cells, including yeast (e.g., S.cerevisiae, S. pombe, P. pastoris, K. lactis, H. polymorpha), and plantcells (Fleer, R. (1992) Current Opinion in Biotechnology 3:486 496).

Nucleases

The methods and compositions described herein are broadly applicable andmay involve any nuclease of interest. Non-limiting examples of nucleasesinclude meganucleases, TAL effector nuclease domain fusion proteins, andzinc finger nucleases. The nuclease may comprise heterologousDNA-binding and cleavage domains (e.g., zinc finger nucleases;TAL-effector nuclease domain fusion proteins, meganuclease DNA-bindingdomains with heterologous cleavage domains) or, alternatively, theDNA-binding domain of a naturally-occurring nuclease may be altered tobind to a selected target site (e.g., a meganuclease that has beenengineered to bind to site different than the cognate binding site).

In certain embodiments, the nuclease is a meganuclease (homingendonuclease). Naturally-occurring meganucleases recognize 15-40base-pair cleavage sites and are commonly grouped into four families:the LAGLIDADG family (“LAGLIDADG” disclosed as SEQ ID NO: 11), theGIY-YIG family, the His-Cyst box family and the HNH family. Exemplaryhoming endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV,I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII andI-TevIII. Their recognition sequences are known. See also U.S. Pat. No.5,420,032; U.S. Pat. No. 6,833,252; Belfort et al. (1997) Nucleic AcidsRes. 25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al:(1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet.12:224-228; Gimble et al. (1996) J. Mol. Biol. 263:163-180; Argast etal. (1998) J. Mol. Biol. 280:345-353 and the New England Biolabscatalogue.

DNA-binding domains from naturally-occurring meganucleases, primarilyfrom the LAGLIDADG family (“LAGLIDADG” disclosed as SEQ ID NO: 11), havebeen used to promote site-specific genome modification in plants, yeast,Drosophila, mammalian cells and mice, but this approach has been limitedto the modification of either homologous genes that conserve themeganuclease recognition sequence (Monet et al. (1999), Biochem.Biophysics. Res. Common. 255: 88-93) or to pre-engineered genomes intowhich a recognition sequence has been introduced (Route et al. (1994),Mol. Cell. Biol. 14: 8096-106; Chilton et al. (2003), Plant Physiology.133: 956-65; Puchta et al. (1996), Proc. Natl. Acad. Sci. USA 93:5055-60; Rong et al. (2002), Genes Dev. 16: 1568-81; Gouble et al.(2006), J. Gene Med. 8(5):616-622). Accordingly, attempts have been madeto engineer meganucleases to exhibit novel binding specificity atmedically or biotechnologically relevant sites (Porteus et al. (2005),Nat. Biotechnol. 23: 967-73; Sussman et al. (2004), J. Mol. Biol. 342:31-41; Epinat et al. (2003), Nucleic Acids Res. 31: 2952-62; Chevalieret al. (2002) Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic AcidsRes. 31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques etal. (2007) Current Gene Therapy 7:49-66; U.S. Patent Publication Nos.20070117128; 20060206949; 20060153826; 20060078552; and 20040002092). Inaddition, naturally-occurring or engineered DNA-binding domains frommeganucleases have also been operably linked with a cleavage domain froma heterologous nuclease (e.g., FokI).

In some embodiments, the nuclease is a TAL-effector domain fusionprotein, where the TAL effector domain is a natural or engineered TALeffector domain fused to a nuclease domain (e.g. FokI). See co-ownedU.S. Patent Application No. 61/395,836 for Novel DNA-binding proteinsand uses thereof, filed May 17, 2010.

In other embodiments, the nuclease is a zinc finger nuclease (ZFN). ZFNscomprise a zinc finger protein that has been engineered to bind to atarget site in a gene of choice and cleavage domain or a cleavagehalf-domain.

Zinc finger binding domains can be engineered to bind to a sequence ofchoice. See, for example, Beerli et al. (2002) Nature Biotechnol.20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan etal. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr.Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct.Biol. 10:411-416. An engineered zinc finger binding domain can have anovel binding specificity, compared to a naturally-occurring zinc fingerprotein. Engineering methods include, but are not limited to, rationaldesign and various types of selection. Rational design includes, forexample, using databases comprising triplet (or quadruplet) nucleotidesequences and individual zinc finger amino acid sequences, in which eachtriplet or quadruplet nucleotide sequence is associated with one or moreamino acid sequences of zinc fingers which bind the particular tripletor quadruplet sequence. See, for example, co-owned U.S. Pat. Nos.6,453,242 and 6,534,261, incorporated by reference herein in theirentireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

Selection of target sites; ZFNs and methods for design and constructionof fusion proteins (and polynucleotides encoding same) are known tothose of skill in the art and described in detail in U.S. PatentApplication Publication Nos. 20050064474 and 20060188987, incorporatedby reference in their entireties herein.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, e.g., U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

Nucleases such as ZFNs, TAL-effector domain nuclease fusions, and/ormeganucleases also comprise a nuclease (cleavage domain, cleavagehalf-domain). As noted above, the cleavage domain may be heterologous tothe DNA-binding domain, for example a zinc finger DNA-binding domain anda cleavage domain from a nuclease or a meganuclease DNA-binding domainand cleavage domain from a different nuclease. Heterologous cleavagedomains can be obtained from any endonuclease or exonuclease. Exemplaryendonucleases from which a cleavage domain can be derived include, butare not limited to, restriction endonucleases and homing endonucleases.See, for example, 2002-2003 Catalogue, New England Biolabs, Beverly,Mass.; and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388.Additional enzymes which cleave DNA are known (e.g., S1 Nuclease; mungbean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HOendonuclease; see also Linn et al. (eds.) Nucleases, Cold Spring HarborLaboratory Press, 1993). One or more of these enzymes (or functionalfragments thereof) can be used as a source of cleavage domains andcleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above; that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any integral number of nucleotides or nucleotidepairs can intervene between two target sites (e.g., from 2 to 50nucleotide pairs or more). In general, the site of cleavage lies betweenthe target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme FokI catalyzes double-strandedcleavage of DNA, at 9 nucleotides from its recognition site on onestrand and 13 nucleotides from its recognition site on the other. See,for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as wellas Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al.(1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc.Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem.269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise thecleavage domain (or cleavage half-domain) from at least one Type IISrestriction enzyme and one or more zinc finger binding domains, whichmay or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is Fok I. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95: 10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the FokI enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-Fok I fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a zinc finger binding domain and two FokI cleavage half-domains can also be used. Parameters for targetedcleavage and targeted sequence alteration using zinc finger-Fok Ifusions are provided elsewhere in this disclosure.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPublication WO 07/014,275, incorporated herein in its entirety.Additional restriction enzymes also contain separable binding andcleavage domains, and these are contemplated by the present disclosure.See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Patent Publication Nos. 20050064474 and 20060188987 andin U.S. application Ser. No. 11/805,850 (filed May 23, 2007), thedisclosures of all of which are incorporated by reference in theirentireties herein. Amino acid residues at positions 446, 447, 479, 483,484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 ofFok I are all targets for influencing dimerization of the Fok I cleavagehalf-domains.

Exemplary engineered cleavage half-domains of Fok I that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFok I and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499.

Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys(K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at486 replaced Gln (Q) with Glu (E); and the mutation at position 499replaces Iso (I) with Lys (K). Specifically, the engineered cleavagehalf-domains described herein were prepared by mutating positions 490(E→K) and 538 (I→K) in one cleavage half-domain to produce an engineeredcleavage half-domain designated “E490K:1538K” and by mutating positions486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce anengineered cleavage half-domain designated “Q486E:1499L”. The engineeredcleavage half-domains described herein are obligate heterodimer mutantsin which aberrant cleavage is minimized or abolished. See, e.g., U.S.Patent Publication No. 2008/0131962, the disclosure of which isincorporated by reference in its entirety for all purposes.

The engineered cleavage half-domains described herein are obligateheterodimer mutants in which aberrant cleavage is minimized orabolished. See, e.g., Example 1 of WO 07/139,898. In certainembodiments, the engineered cleavage half-domain comprises mutations atpositions 486, 499 and 496 (numbered relative to wild-type FokI), forinstance mutations that replace the wild type Gln (Q) residue atposition 486 with a Glu (E) residue, the wild type Iso (I) residue atposition 499 with a Leu (L) residue and the wild-type Asn (N) residue atposition 496 with an Asp (D) or Glu (E) residue (also referred to as a“ELD” and “ELE” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490,538 and 537 (numbered relative to wild-type FokI), for instancemutations that replace the wild type Glu (E) residue at position 490with a Lys (K) residue, the wild type Iso (I) residue at position 538with a Lys (K) residue, and the wild-type His (H) residue at position537 with a Lys (K) residue or a Arg (R) residue (also referred to as“KKK” and “KKR” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490 and537 (numbered relative to wild-type FokI), for instance mutations thatreplace the wild type Glu (E) residue at position 490 with a Lys (K)residue and the wild-type His (H) residue at position 537 with a Lys (K)residue or a Arg (R) residue (also referred to as “KIK” and “KIR”domains, respectively). (See U.S. provisional application 61/337,769filed Feb. 8, 2010).

Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (Fok I) as described in U.S. PatentPublication Nos. 20050064474 and 20080131962.

Alternatively, nucleases may be assembled in vivo at the nucleic acidtarget site using so-called “split-enzyme” technology (see e.g. U.S.Patent Publication No. 20090068164). Components of such split enzymesmay be expressed either on separate expression constructs, or can belinked in one open reading frame where the individual components areseparated, for example, by a self-cleaving 2A peptide or IRES sequence.Components may be individual zinc finger binding domains or domains of ameganuclease nucleic acid binding domain.

Nucleases (e.g., ZFNs) can be screened for activity prior to use, forexample in a yeast-based chromosomal system as described in WO2009/042163 and 20090068164.

Nuclease expression constructs can be readily designed using methodsknown in the art. See, e.g., United States Patent Publications20030232410; 20050208489; 20050026157; 20050064474; 20060188987;20060063231; and International Publication WO 07/014,275. Expression ofthe nuclease may be under the control of a constitutive promoter or aninducible promoter, for example the galactokinase promoter which isactivated (de-repressed) in the presence of raffinose and/or galactoseand repressed in presence of glucose.

Cold Shock Conditions

The methods described herein involve subjecting the host cells to aperiod of cold shock before, during or after introduction of thenuclease(s) and/or donor polynucleotide. Typically, the cells arecold-shocked following introduction of (e.g., transfection with) thenuclease and/or donor nucleotide. The cells may be cold-shocked withinminutes after transfection or may be maintained at 37° C. for a shortperiod of time (1 day for example) prior to shifting to the coolertemperature.

The period of time for which the cells are cold shocked can vary fromhours to days. In certain embodiments, the cells are cold-shocked forbetween 1 and 4 days. It will be apparent that the period of cold shockwill also vary depending on the cell type into which the nuclease isintroduced.

Likewise, the temperature at which the cells are cold-shocked is anytemperature that reduces cell division, but at which the nuclease(s) is(are) expressed and/or active. Suitable temperatures will vary dependingon the host cell type. For mammalian cells, cold shock temperaturesinclude, but are not limited to, 33° C., 32° C., 31° C., 30° C., 29° C.,28° C., 27° C., and even lower. For zebrafish and plant cells, the coldshock temperatures will typically be lower, for example 26° C., 25° C.,24° C., 23° C., 22° C., 21° C., 20° C., 19° C., 18° C. or event lower.Furthermore, the temperature can vary during the period ofcold-shocking, so long as it remains low enough so that the cells arenot dividing or are dividing at a reduced rate.

Kits

Also provided are kits for performing any of the above methods. The kitstypically contain polynucleotides encoding one or more nucleases and/ordonor polynucleotides as described herein as well as instructions forcold-shocking the cells into which the nucleases and/or donorpolynucleotide are introduced. The kits can also contain cells, buffersfor transformation of cells, culture media for cells, and/or buffers forperforming assays. Typically, the kits also contain a label whichincludes any material such as instructions, packaging or advertisingleaflet that is attached to or otherwise accompanies the othercomponents of the kit.

Applications

The disclosed methods and compositions can be used for increasingbiological activity of nucleases on their targets in the genome of anyhost cell. Typically, the host cells are first transfected with anexpression construct that directs expression of one or more nucleaseswithin the cell. Following transfection, the cells are placed in a coldshock condition that allows for expression of the nucleases but in whichthe rate of cell division is reduced. Following the period of coldshock, the cells are returned to a temperature suitable for increasedcell division. Under these conditions, the efficiency of gene disruptionis greatly increased, and, accordingly, the methods described hereinallow for the rapid generation of knock out cell lines (cells in whichone or more genes have been deleted (“knocked out”) or disrupted).Similarly, the methods described herein facilitate the generation ofcell lines wherein one or more genes or nucleic acids have beenintroduced into the genome.

In addition, the compositions and methods described herein allow forefficient generation of primary cells containing nuclease-modifiedgenomes. Nuclease-modified cells may be used in therapeuticapplications, for example by deleting a receptor for a virus (see UnitedStates Publication 20080159996) or for a growth factor (see UnitedStates Publication 20080188000). Such cells may be then re-introducedinto a mammal to carry out a therapeutic effect.

Further, the methods and compositions described herein may be used inplant cells. Plant cells carrying genomic modifications created by thecontemplated methods and compositions may be used to regenerate wholeplants and create novel plant lines. Increasing nuclease activity maylead to increased ability to generate plant lines with multipleintroduced desired traits (i.e. trait stacking).

Additionally, methods and compositions described herein may be used inthe construction of transgenic animals, Genomic modifications (via NHEJor additions and/or deletions) may be introduced in embryos, and thenthese genomically modified embryos may be used to create transgenicanimals using any known suitable method.

Methods and compositions described herein are also used in kits suitablefor the optimization of nucleases as well as for targeted nucleic acidinsertion or deletion into the genome of a cell.

The following Examples relate to exemplary embodiments of the presentdisclosure in which the nuclease comprises a zinc finger nuclease (ZFN).It will be appreciated that this is for purposes of exemplification onlyand that other nucleases can be used, for instance homing endonucleases(meganucleases) with engineered DNA-binding domains and/or fusions ofnaturally occurring of engineered homing endonucleases (meganucleases)DNA-binding domains and heterologous cleavage domains or TAL-effectordomain nuclease fusion proteins.

EXAMPLES Example 1 Preparation of ZFNs

ZFNs targeted to the various genes used in this study were designed andincorporated into plasmids or adenoviral vectors essentially asdescribed in Urnov et al. (2005) Nature 435(7042):646-651, Perez et al(2008) Nature Biotechnology 26(7): 808-816, and U.S. Patent Publication20080299580: Zinc finger proteins were designed as described in co-ownedU.S. Pat. Nos. 6,453,242 and 6,534,261. See Table 1 for the amino acidsequences of the recognition regions of the AAVS1 zinc finger proteinsand Table 2 for the sequences of the target sites. Sequences encodingeach of these two ZFP binding domains were fused to sequences encoding aFokI cleavage half-domain (amino acids 384-579 of the native FokIsequence; Kita et al. (1989) J. Biol. Chem. 264:5751-5756), such thatthe encoded protein contained FokI sequences at the carboxy terminus andZFP sequences at the amino terminus.

TABLE 1 Zinc Finger helices used Gene/ ZFN name F1 F2 F3 F4 AAVS1/QSSNLAR RTDYLVD YNTHLTR QGYNLAG 15590 (SEQ ID NO: 1) (SEQ ID NO: 2)(SEQ ID NO: 3) (SEQ ID NO: 4) AAVS1/ YNWHLQR RSDHLTT HNYARDC QNSTRIG15556 (SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) (SEQ ID NO: 8) Note:The zinc finger amino acid sequences shown above (in one-letter code)represent residues −1 through +6, with respect to the start of thealpha-helical portion of each zinc finger. Finger F1 is closest to theamino terminus of the protein, and Finger F4 is closest to the carboxyterminus.

TABLE 2 ZFN Target sequences Gene/ ZFN name Target Site AAVS1/acTAGGGACAGGATtg 15590 (SEQ ID NO: 9) AAVS1/ ccCCACTGTGGGGTgg 15556(SEQ ID NO: 10)

Additional ZFNs targeted to the following genes were also obtained fromSigma-Aldrich: KDR, TP73, MAP3K14, EP300, BTK135, CARM1, GNAI2, TSC2,RIPK1, and KDR. GR-targeted ZFNs were prepared as described above and inU.S. Patent Publication No. 2008/0188000.

Example 2 Increased Nuclease Activity Following Cold Shock

ZFN activity was increased several fold when cells were treated with acold shock condition following transfection. Briefly, the plasmidencoding ZFP-FokI fusions were introduced into K562 cells bytransfection using the Amaxa™ Nucleofection kit as specified by themanufacturer. For transfection, two million K562 cells were mixed withvarying amounts of each zinc-finger nuclease expression plasmid and 100μL Amaxa Solution V. Cells were transfected in an Amaxa Nucleofector II™using program T-16. Immediately following transfection, the cells weredivided into two different flasks and grown in RPMI medium (Invitrogen)supplemented with 10% FBS in 5% CO₂ at either 30° C. or 37° C. for fourdays. To determine the ZFN activity at the appropriate locus (e.g.,AAVS1 locus for AAVS1 targeted ZFPs), CEL-I mismatch assays wereperformed essentially as per the manufacturer's instructions(Trangenomic SURVEYOR™).

Cells were harvested and chromosomal DNA prepared using a Quickextract™Kit according to manufacturer's directions (Epicentre®). The appropriateregion of the target locus was PCR amplified using Accuprime™High-fidelity DNA polymerase (Invitrogen). PCR reactions were heated to94° C., and gradually cooled to room temperature. Approximately 200 ngof the annealed DNA was mixed with 0.33 μL CEL-I enzyme and incubatedfor 20 minutes at 42° C. Reaction products were analyzed bypolyacrylamide gel electrophoresis in 1× Tris-borate-EDTA buffer.

Nuclease activity, as measured by CEL-I detection of NHEJ activity,increased in the cells that had been incubated at 30° C. FIG. 1A showsresults using AAVS1-targeted ZFNs. Control lanes from experiments using0 μg of ZFN expression plasmid showed no mismatches, while theexperiment that used 0.5 μg ZFN expression plasmid showed a largeincrease in the percent of modification. The same was true forexperiments using 1 and 2.5 μg of input ZFN expression plasmid.Furthermore, at 30° C. cold-shock incubation, all results were in thenon-linear range of detection (above approximately 40% mismatch, asdenoted by an *), indicating that the assay was saturated at this highof a percentage of mismatch.

FIG. 2 shows results using MAP3K14, EP300, BTK, CARM1 and GNAI2-targetedZFNs. In all cases, cold shock conditions (FIG. 2B) enhanced. ZFNactivity (2 to 10 fold or more) as compared to no cold shock (FIG. 2C),as measured by the increased frequency of genomic modificationsfollowing ZFN cleavage. Importantly, ZFNs pairs (MAP3K14 and CARM1) forwhich activity was not detected at 37° C. were sufficiently activefollowing cold shock to provoke a robust gene disruption signal (FIG.2B).

Cold shock treatment also increased ZFN activity for all ZFNs tested atsub-maximal DNA vector doses, with the increase in CEL-I signaldemonstrating stability over time in culture.

In addition, ZFN protein level and activity under normal and cold shockconditions was also determined. Using ZFNs targeting the KDR gene, adose-dependent and stable augmentation in CEL-I signal with increasingvector concentration, an effect enhanced by the use of an introncontaining vector at 37° C. Western blotting revealed a marked increasein the steady state level of ZFN protein under all cold shock conditionstested, an effect that paralleled the improvement in gene disruptionefficiency in both HeLa and K562 cells. Thus, the increase in ZFNactivity obtained via a transient cold shock results, at least in part,from the accumulation of ZFN protein.

Example 3 Increase in ZFN Activity is not Cell Type Specific andModifications are Stable Over Time

To examine if the increase in ZFN activity could be observed for morethan one cell type, the experiments were repeated with HeLa cells. Cellswere transfected with 0.1, 0.2 or 0.4 μg of AAVS1-specific ZFNs usingthe 96-well Nucleofector® Kit SE as per manufacturer recommendations. Inaddition, cells were incubated for 3 days following transfection at the30° C. or 37° C. incubation temperatures.

As shown in FIG. 3A, control experiments without added ZFN expressionplasmid (lane labeled “0”) did not exhibit any mismatches while cellstransfected with 0.1, 0.2 or 0.4 μg of expression plasmid did. Again,large increases in nuclease activity were seen in the cells incubated at30° C. as compared to those incubated at 37° C. As was seen in Example2, the NHEJ values determined for those cells incubated at 30° C. werein the non-linear range of the assay (indicated by *) as the assaybecame oversaturated. Similarly, FIG. 4A shows an increase in ZFNactivity following cold shock using a KDR-targeted ZFN (Sigma-Aldrich).

To determine if the modifications are stable over time, a portion ofpopulations of the cells were maintained at 37° C. or 30° C. for anadditional cell doublings. Following additional time in culture, cellswere then processed for the CEL-I assay as above.

Exemplary results are shown in FIGS. 3B and 4B. The data indicates thatthe amount of measured nuclease activity, as assayed by NHEJ activity,is very similar to that measured following the initial incubationperiod. Thus, the increase in genomic modification due to increasednuclease activity is not lost during the 37° C. incubation following theinitial 30° C. incubation.

Example 4 Summary of Data Obtained from Numerous Cell Types

The method as described above was repeated with several different celltypes, using varying methods of transfection and various ZFNs. Detailsabout the transfection methods used are as follows. For transfection ofZFN expression plasmids via electroporation, the methodology used wasessentially as above. For transduction of cells with a lentiviral basedZFN expression vector, the methodology used was as described in Lombardoet al, (2007) Nature Biotechnology vol 25 (11): 1209-1306.

In this survey analysis, two different types of nuclease domains in theZFNs were used as well, the wild type domain (wt) as well as theobligate heterodimeric domain (‘HiFi’). Cell types used were from human,mouse, rat hamster and pig origins. As is seen in Table 3 below, theincrease in nuclease activity observed from the 30° C. treatment variedfrom a 1.5 to a 15-fold increase in activity as compared to 37° C.alone. In all cases shown, the cells received a 3 day 30° C. cold shockprior to analysis. Overall, the largest increases in nuclease activitywere observed in ZFN with the lowest amounts of activity and with theZFNs wherein the nuclease domain was of the obligate heterodimeric type.

TABLE 3 Summary of cell lines tested for ZFN activity under cold shockconditions # ZFN Fold pairs FokI Transfection Species Cell type effecttested domain method Human K562 1.5*-5x  7 wt, HiFi electroporationHuman Hela    2->15x 7 HiFi electroporation Human HEK 293 1.5*-15x 4 wt,HiFi lipofection electroporation Mouse Splenocytes 2x 1 wt, HiFielectroporation Rat C6 4x 1 HiFi lentivirus Hamster CHO-K1  1.5^(†)-2*x3 wt, HiFi electroporation Pig PK15   3-12x 4 HiFi electroporation*These ZFN produce >15% NHEJ at 37° C. ^(†)These ZFN produce >20% NHEJat 37° C.

Thus, cold shock resulted in similar improvements in primary cells aswell as transformed lines derived from a variety of species, independentof the ZFN pair or delivery method.

In addition, nuclease activity was also tested following differentperiods of cold shock (e.g., 2 days, 3 days). While cold shockconditions increased nuclease activity, optimal treatment durationvaried among cell lines.

Example 5 Preferentially Cleavage of Target Sequences Under Cold ShockConditions

To determine whether increased ZFN protein levels alter specificity, theratio was between on-target and off-target cleavage for a humanglucocorticoid receptor gene (GR) targeted ZFN for which two off-targetcleavage sites were known in K562 cells (both in non-coding regions ofthe genome) was determined as described above by CEL-1 assay. Inparticular, K652 cells were nucleofected with a GFP expression plasmid(−) or 80 ng of a CMV promoter-driver ZFN expression vector (lanes 2-5and 7-10) targeted to the GR gene containing either wild-type FokIcleavage domains or obligate heterodimer FokI cleavage domains.Immediately after transfection, cells were divided and incubated for 3days at 37° C. or 3 days at 30° C. The frequency of indels at the GRlocus as well as off-target sites Trim26 and chromosome 1 were assessedby CEL-1 assay 3 days post-transfection.

As shown in FIG. 5, higher levels of ZFN protein (via cold shock)resulted in a proportional increase in ZFN activity at the intended (GR)and both off-target sites (FIG. 5). In contrast, the increase inoff-target modification was markedly lower, yet on-target modificationvery high, when obligate heterodimer Fold variants were used. These datashow that cold shock conditions can increase both activity andspecificity of nucleases.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entirety.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

What is claimed is:
 1. A method for increasing the nuclease activity ofat least one zinc finger nuclease in an isolated mammalian cell, themethod comprising: (a) culturing the isolated mammalian cell at anoptimal growth temperature above 33° C.; (b) transfecting the isolatedmammalian cell with a polynucleotide encoding the at least one zincfinger nuclease (ZFN) into the isolated mammalian cell from step (a);(c) culturing the isolated mammalian cell from step (b) at a temperaturebetween 27° C. and 33° C. for between 1 and 4 days; and (d) culturingthe isolated mammalian cell following step (c) at the optimal growthtemperature, such that the nuclease activity of the ZFN is increased ascompared to an isolated mammalian cell transfected and cultured only atthe optimal growth temperature without the culturing step (c).
 2. Themethod of claim 1, wherein the polynucleotide is transfected into theisolated mammalian cell using a viral vector, a plasmid or an RNA. 3.The method of claim 2, wherein the viral vector is an IntegrationDefective Lentiviral vector (IDLV) construct.
 4. The method of claim 1,further comprising introducing an exogenous donor nucleic acid sequenceinto the isolated mammalian cell, wherein the isolated mammalian cellcomprises a genome and the exogenous donor nucleic acid sequence isintegrated into the genome of the isolated mammalian cell.